CDC7 IS REQUIRED THROUGHOUT THE YEAST S-PHASE TO ACTIVATE REPLICATIONORIGINS

The long-standing conclusion that the Cdc7 kinase of Saccharomyces cer evisiae is required only to trigger S phase has been challenged by rec ent data that suggests it acts directly on individual replication orig ins. We tested the possibility that early-and late-activated origins h ave different requirements for Cdc7 activity. Cells carrying a cdc7(ts ) allele were first arrested in G(1) at the cdc7 block by incubation a t 37 degrees C, and then were allowed to enter S phase by brief incuba tion at 23 degrees C. During the S phase, after return to 37 degrees C , early-firing replication origins were activated, but late origins fa iled to fire. Similarly, a plasmid with a late-activated origin was de fective in replication. As a consequence of the origin activation defe ct, duplication of chromosomal sequences that are normally replicated from late origins was greatly delayed. Early-replicating regions of th e genome duplicated at approximately their normal time. The requiremen ts of early and late origins for Cdc7 appear to be temporally rather t han quantitatively different, as reducing overall levels of Cdc7 by gr owth at semi-permissive temperature reduced activation at early and la te origins approximately equally. Our results show that Cdc7 activates early and late origins separately, with late origins requiring the ac tivity later in S phase to permit replication initiation.